The present invention relates to a protein which catalyzes the synthesis of a dipeptide, a process for producing the protein which catalyzes the synthesis of a dipeptide, a process for producing a dipeptide using the protein which catalyzes the synthesis of a dipeptide, a microorganism or a transformant producing the protein which catalyzes the synthesis of a dipeptide, and a process for producing the dipeptide using such microorganism or transformant.
Chemical synthesis methods (liquid phase method and solid phase method), enzymatic synthesis methods and biological synthesis methods utilizing recombinant DNA techniques are available for large-scale peptide synthesis. Currently, the enzymatic synthesis methods and biological synthesis methods are employed for the synthesis of long-chain peptides longer than 50 residues, and the chemical synthesis methods and enzymatic synthesis methods are mainly employed for the synthesis of dipeptides.
In the synthesis of dipeptides by the chemical synthesis methods, however, operations such as introduction and removal of protective groups for functional groups are necessary, and racemates are also formed. The chemical synthesis methods are thus considered to be disadvantageous in respect of cost and efficiency. They are unfavorable also from the viewpoint of environmental hygiene because of the use of large amounts of organic solvents and the like.
As to the synthesis of dipeptides by the enzymatic methods, the following methods are known: a method utilizing reverse reaction of protease (J. Biol. Chem., 119, 707-720 (1937)); methods utilizing thermostable aminoacyl t-RNA synthetase (Japanese Published Unexamined Patent Application No. 146539/83, Japanese Published Unexamined Patent Application No. 209991/83, Japanese Published Unexamined Patent Application No. 209992/83,and Japanese Published Unexamined Patent Application No. 106298/84); and methods utilizing non-ribosome peptide synthetase (hereinafter referred to as NRPS) (Chem. Biol., 7, 373-384 (2000), FEBS Lett., 498, 42-45 (2001), U.S. Pat. No. 5,795,738 and U.S. Pat. No. 5,652,116).
However, the method utilizing reverse reaction of protease requires introduction and removal of protective groups for functional groups of amino acids used as substrates, which causes difficulties in raising the efficiency of peptide-forming reaction and in preventing peptide-degradating reaction. The methods utilizing thermostable aminoacyl t-RNA synthetase have the defects that the expression of the enzyme and the prevention of side reactions forming by-products other than the desired products are difficult. The methods utilizing NRPS are inefficient in that the expression of the enzyme by recombinant DNA techniques is difficult because of its large enzyme molecule size and that the supply of coenzyme 4′-phosphopantetheine is necessary.
On the other hand, there exist a group of peptide synthetases that have enzyme molecular weight lower than that of NRPS and do not require coenzyme 4′-phosphopantetheine; for example, γ-glutamylcysteine synthetase, glutathione synthetase, D-alanine-D-alanine (D-Ala-D-Ala) ligase, and poly-γ-glutamate synthetase. Most of these enzymes utilize D-amino acids as substrates or catalyze peptide bond formation at the γ-carboxyl group. Because of such properties, they can not be used for the synthesis of dipeptides by peptide bond formation at the α-carboxyl group of L-amino acid.
The only known example of an enzyme capable of dipeptide synthesis by the activity to form a peptide bond at the α-carboxyl group of L-amino acid is bacilysin (dipeptide antibiotic derived from a microorganism belonging to the genus Bacillus ) synthetase. Bacilysin synthetase is known to have the activity to synthesize bacilysin [L-alanyl-L-anticapsin (L-Ala-L-anticapsin)] and L-alanyl-L-alanine (L-Ala-L-Ala), but there is no information about its activity to synthesize other peptides (J. Ind. Microbiol., 2, 201-208 (1987) and Enzyme. Microbial. Technol., 29, 400-406 (2001)).
As for the bacilysin biosynthetase genes in Bacillus subtilis 168 whose entire genome information has been clarified (Nature, 390, 249-256 (1997)), it is known that the productivity of bacilysin is increased by amplification of bacilysin operons containing ORFs ywfA-F (WO00/03009 pamphlet). However, it is not known whether an ORF encoding a protein having the activity to ligate two or more amino acids by peptide bond is contained in these ORFs, and it contained, which ORF encodes the protein.
An object of the present invention is to provide a protein which catalyzes the synthesis of a dipeptide which is different from L-Ala-L-Ala and for which no enzymatic synthesis method using peptide synthetase or the like has so far been proposed, and a protein for the synthesis of the dipeptide; DNA encoding the protein having the dipeptide-synthesizing activity; a recombinant DNA comprising the DNA; a transformant carrying the recombinant DNA; a process for producing the protein having the dipeptide-synthesizing activity; an enzymatic method for synthesizing the dipeptide using the protein having the dipeptide-synthesizing activity or the protein for the dipeptide synthesis; and a process for producing the dipeptide using, as an enzyme source, a culture of a microorganism having the ability to produce the protein having the dipeptide-synthesizing activity or the protein for the dipeptide synthesis, or the like.
An object of the present invention is to provide a protein which catalyzes the synthesis of a dipeptide which is different from L-Ala-L-Ala and for which no enzymatic synthesis method using peptide synthetase or the like has so far been proposed, and a protein for the synthesis of the dipeptide; DNA encoding the protein having the dipeptide-synthesizing activity; a recombinant DNA comprising the DNA; a transformant carrying the recombinant DNA; a process for producing the protein having the dipeptide-synthesizing activity; an enzymatic method for synthesizing the dipeptide using the protein having the dipeptide-synthesizing activity or the protein for the dipeptide synthesis; and a process for producing the dipeptide using, as an enzyme source, a culture of a microorganism having the ability to produce the protein having the dipeptide-synthesizing activity or the protein for the dipeptide synthesis, or the like.